1. Field of the Invention
The present invention relates to methods of identifying agents that upregulate CTLA-4 that are useful in immunosuppression and the generation of immunologic tolerance for the prevention and treatment of transplant rejection and autoimmune and inflammatory diseases.
2. Description of the Related Art
Transplantation is now the treatment of choice for end-stage heart, kidney, and liver disease. Although improved immuno-suppression has led to excellent short-term allograft survival, acute rejection still occurs and long-term results remain inadequate. Moreover, sub-clinical rejection is still relatively frequent on protocol biopsies and may contribute to chronic rejection. Finally, current therapy requires life-long immunosuppression with attendant risks of infection and malignancy. Therefore, there is a need to develop improved immunosuppressive agents that are both more effective and more specific for prevention of rejection (with less generalized immunosuppression and side-effects). The ideal therapy would consist of a finite course of treatment that would induce specific tolerance (lack of responsiveness) for the transplant, while leaving the immune system intact to defend against other threats. Achieving tolerance would reduce rejection, increase long-term engraftment, and eliminate continuous immunosuppression, thereby reducing morbidity, mortality, and cost.
Under normal circumstances, the immune system exhibits tolerance (i.e. lack of responsiveness) to self-antigens. Abnormalities in self-tolerance lead to immune responses against self and debilitating inflammatory disorders commonly called autoimmune diseases. These include rheumatoid arthritis, type I diabetes, systemic lupus erythematosis, inflammatory bowel disease (such as ulcerative colitis and Crohn's disease), myasthenia gravis, multiple sclerosis, vitiligo, pernicious anemia, among many others. As in the case of transplantation, current therapy has variable success and is fraught with risks of over-immunosuppression (malignancy, infection) and other side effects (cardiovascular disease, hypertension, diabetes). Once again, more effective immunomodulatory agents, particularly those able to restore immunologic tolerance, would be of great benefit.
The mechanisms underlying tolerance are complex and poorly understood, but it is clear that T lymphocytes, commonly called T cells, play a primary role in both allograft rejection and autoimmunity. T cell activation is not “all or none”. Rather, the T cell response upon exposure to antigen can be modified, resulting in downregulation, anergy, or the development of regulatory cells which inhibit the activity of other T cells.
CTLA-4 (Cytotoxic T cell Antigen-4; CD 152) is a molecule expressed primarily by T lymphocytes that is critically involved in downregulation of the immune response and in the generation of tolerance (specific non-responsiveness) in transplantation and autoimmunity. CTLA-4 normally interacts with a ligand on antigen presenting cells called B7 (CD80 and CD86)—producing an inhibitory signal. Animals genetically deficient in CTLA-4 rapidly develop a uniformly fatal autoimmune syndrome. In normal animals, blockade of CTLA-4-mediated negative signaling with antibodies results in augmented immune responsiveness against tumors and prevents tolerance to transplanted organs, soluble antigens, and can precipitate de novo autoimmune disease. Such data indicate that augmentation of CTLA-4-derived negative signals would be of great help in promoting stable engraftment of transplanted tissues and in preventing/treating autoimmunity. Unfortunately there are no known agonist ligands to CTLA-4 that will act in soluble form. All known antibodies against CTLA-4 only block the CTLA-4 signal and actually augment immune responsiveness as noted above. Anti-CTLA-4 mAbs only give rise to inhibitory signals when they are extensively cross-linked (e.g., on plastic or rubber beads, or by co-administration of an another antibody that recognizes the anti-CTLA-4 (anti-globulin)). These are not practical approaches in vivo.
CTLA-4 expression is tightly controlled, however, and little is known about specific signals involved in upregulating its expression. Normally expression is upregulated only after a cell is fully activated and enters the cell cycle, reaching maximal levels 72 hours after ligation of the T cell receptor and CD28 costimulatory molecules. CTLA-4 expression is primarily intracellular. However, cell activation induces rapid cycling to the cell surface and back. How constitutive CTLA-4 expression (i.e., small amounts that are present prior to activation) is regulated is completely unknown. Recently it has become clear that cells that express even low levels of CTLA-4 may play a critical role as regulatory cells that down-regulate autoimmune responses (thereby preventing autoimmune disease). Such cells may also play an important role in preventing rejection and promoting transplant tolerance. However, until now there has been no known way to capitalize on this potent downregulatory pathway.
In summary, animals genetically deficient in CTLA-4 rapidly develop a uniformly fatal lymphoproliferative/autoimmune syndrome. In normal animals, blockade of CTLA-4-mediated negative signaling with antibodies, results in augmented immune responsiveness against tumors and prevents tolerance to transplanted organs or soluble antigens. Such data indicate that augmentation of CTLA-4-derived signals would be of great help in promoting stable engraftment of transplanted tissues and in preventing/treating autoimmunity. Unfortunately, at the present time, there are no candidate antibodies, soluble ligands, or small molecules that augment CTLA-4-mediated negative signals. Another approach, equally unfeasible until now, would be to specifically upregulate CTLA-4 expression, allowing for augmented CTLA-4-signaling through interaction with its physiological B7 ligands. It would be very useful to have screens to identify agents that act by either means to augment CTLA-4 signaling.